Integration of Metabolome and Transcriptome Profiling Reveals the Effect of Modified Atmosphere Packaging (MAP) on the Browning of Fresh-Cut Lanzhou Lily (Lilium davidii var. unicolor) Bulbs during Storage

The fresh-cut bulbs of the Lanzhou lily (Lilium davidii var. unicolor) experience browning problems during storage. To solve the problem of browning in the preservation of Lanzhou lily bulbs, we first investigated the optimal storage temperature and gas ratio of modified atmosphere packaging (MAP) of Lanzhou lily bulbs. Then, we tested the browning index (BD), activity of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD), the content of malonaldehyde (MDA) and other physiological activity indicators related to browning. The results showed that the storage conditions of 10% O2 + 5% CO2 + 85% N2 and 4 °C were the best. To further explore the anti-browning mechanism of MAP in fresh-cut Lanzhou lily bulbs, the integration of metabolome and transcriptome analyses showed that MAP mainly retarded the unsaturated fatty acid/saturated fatty acid ratio in the cell membrane, inhibited the lipid peroxidation of the membrane and thus maintained the integrity of the cell membrane of Lanzhou lily bulbs. In addition, MAP inhibited the oxidation of phenolic substances and provided an anti-tanning effect. This study provided a preservation scheme to solve the problem of the browning of freshly cut Lanzhou lily bulbs, and discussed the mechanism of MAP in preventing browning during the storage of the bulbs.


Introduction
The Lanzhou lily (Lilium davidii var. unicolor) is a perennial herb of the liliaceae family and the bulb is its main edible part. It is mainly distributed in Lanzhou city, Gansu Province, China. According to local government statistics, the planting area of the Lanzhou lily has reached over 700,000 square meters in Lanzhou, and annual production has exceeded 20,000 tons. It has high pharmacological activity and nutritional value; not only does it have polysaccharides, fats, protein, crude fiber, vitamins and other nutrients, but it also contains saponins, alkaloids, flavonoids and other active ingredients in its bulb [1]. Lanzhou lily polysaccharides are known to have anti-inflammation, anti-tumor, and anti-oxidant functions [2,3].
In recent years, with increasing demand for edible, healthy fruits and vegetables, the global market for fresh-cut fruits and vegetables (FFV) has grown rapidly. FFV refers to fresh fruits and vegetables as raw materials after the cleaning, selecting, cutting, molding, preserving and packaging processing of healthy, high-nutrition fruit and vegetable products for convenient consumption [4]. There are also great problems in the production process of FFV, including how to solve tissue softening, cutting surface browning, corruption and other

Determination of BD
According to method of Yang et al. [17], with slight modifications, we weighed 2.0 g of lily sample, added it to a pre-cooled mortar, added distilled water at 0 • C to 4 • C at a ratio of 1:5 (w/v), then added a small amount of quartz sand under ice bath conditions to quickly grind it into a uniform size. The slurry was centrifuged at 4 • C and 5000 r/min for 10 min, and the supernatant was taken at a wavelength of 420 nm to measure the absorbance A (ultraviolet spectrophotometer, UV-1000, Labtech, Boston, MA, USA), with distilled water as the blank control. The results were expressed as 10 × A420 for the BD of Lanzhou lily.
BD was determined by following the method of Kan et al. [18] with slight modifications; lily bulb tissue was homogenized at 4 • C using distilled water and centrifuged (5000 r/min (931M-1029-0, Beckman, Brea, CA, USA), 10 min). The absorbance value at 420 nm was determined by a spectrophotometer (Ultraviolet spectrophotometer, UV-1000, Labtech, USA) and BD was expressed as A420 × 10.

Extraction and Assay of PPO, POD and PAL
Lanzhou lily bulb tissue (2.0 g) was homogenized at 4 • C with cold extraction buffer containing phosphate buffer (0.05 mol/L, pH 6.0) and 2% polyvinylpolypyrrolidone (PVPP), and centrifuged (4 • C, 12,000 r/min, 15 min) for the assay of the activities of PPO. Lily bulb tissue (2.0 g) was homogenized at 4 • C with cold extraction buffer containing phosphate buffer (0.05 mol/L, pH 8.0) and 2% polyvinylpolypyrrolidone (PVPP). The homogenates were then centrifuged at 12,000 r/min for 15 min at 4 • C for the assay of the activities of POD. Lily bulb tissue (2.0 g) was homogenized at 4 • C using boric acid-borax buffer (0.1 mol/L, pH 8.8), 1 mmol/L EDTA, 1% polyvinylpyrrolidone PVP and 20 mmol/L mercaptoethanol, then centrifuged (4 • C, 12,000 r/min, 20 min) for the assay of the activities of PAL.
PPO and POD activity was assayed according to method of Yingsanga et al. [19], with slight modifications. For POD activity measure, 0.5 mL of supernatant was mixed with 1.5 mL of phosphate buffer (0.1 mol/L, pH 6.8) and 1 mL catechol solution (0.1 mol/L). Change in absorbance at 410 nm was measured spectrophotometrically. An enzyme activity unit (U) was defined spectrophotometrically as an increase of 0.01 in absorbance per minute per milliliter. For POD activity analysis, 0.5 mL of enzyme extract was added to a reaction mixture consisting of 0.1 mol/L phosphate buffer (pH 8.0), 0.05 mol/L guaiacol solution and 0.5 mol/L H 2 O 2 (2.5 mL). The change of the mixtion in absorbance at 470 nm was recorded once every 30 s. One unit (U) was defined spectrophotometrically as an increase of 0.01 in absorbance per minute per gram.
PAL activity was assayed according to method of Assis et al. [20], with slight modifications. An amount of 0.5 mL of enzyme extract was added to a reaction mixture consisting of 0.1mol/L boronate-borax buffer solution (PH = 8.8) (3.5 mL) and 20 mmol/L L-phenylalanine (1 mL), then placed in a 40 • C water bath for 50 min. After the incubation, 6 mol/L hydrochloric acid solution (0.1 mL) was added to terminate the reaction immedi- ately. The change of the mixtion in absorbance at 290 nm was recorded. One unit (U) was defined spectrophotometrically as an increase of 0.01 in absorbance per hour per gram.

Determination of Malondialdehyde (MDA)
MDA was assayed according to method of Liu et al. [21], with slight modifications. Lily bulb tissue (2.0 g) was homogenized at 4 • C with cold extraction buffer containing trichloroacetic acid and centrifuged (4 • C, 10,000 r/min, 20 min). For the assay of MDA, 2.0 mL of supernatant was mixed with 2.0 mL 0.67% thiobarbituric acid. Change in absorbance at 532 nm, 600 nm and 450 nm were measured spectrophotometrically.
Total RNA of each sample was extracted using TRIzol Reagent/RNeasy Mini Kit (Qiagen)/other kits. Total RNA of each sample was quantified and qualified by Agilent 2100/2200 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc., Waltham, MA, USA). An amount of 1 µg total RNA was used for following library preparation. In order to remove technical sequences, including adapters, polymerase chain reaction (PCR) primers, or fragments thereof, and quality of bases lower than 20, pass filter data of fastq format were processed by Cutadapt (v1.9.1) to become high-quality, clean data. Differential expression analysis used the DESeq2 Bioconductor package (v1.6.3), p-adjusted (Padj) of genes was set <0.05 to detect differentially expressed genes. GOSeq (v1.34.1) was used to identify Gene Ontology (GO) terms. KEGG (Kyoto Encyclopedia of Genes and Genomes) was used to enrich significant differential expression genes in KEGG pathways.

Metabolite Profiling and Data Analysis
The samples from the 1d (bx (MAP)1, ck(control)1), 4d (bx2, ck2), 8d (bx3, ck3) and 14d (bx4, ck4) of the 10% O 2 + 5% CO 2 + 85% N 2 modified atmosphere preservation experiment at 4 • C were selected, and the samples were kept at a low temperature of 4 • C during the same period as the control academic analysis. There were six replicate samples per group.
Preparation of sample for Liquid chromatography-Mass spectrometer/Mass spectrometer (LC-MS/MS), the Lanzhou lily bulb samples were frozen with liquid nitrogen and pulverized into powder. Pre-cooled acetonitrile (Cat. No. 271004, Sigma-Aldrich, St. Louis, MO, USA): methanol (Cat. No. 34860, Sigma-Aldrich, St. Louis, MO, USA): water = 2:2:1 (v/v/v) was vortexed and ultra-sonicated at low temperature for 30 min, incubated at −20 • C for 10 min, centrifuged at 4 • C for 20 min at 14,000× g, and the supernatant was vacuum dried before LC-MS/MS analysis. Samples were kept at −80 • C. We added 150 mL of acetonitrile aqueous solution (acetonitrile: water = 1:1 v/v) to each sample. The mixture was then vortexed for 1 min before centrifugation at 14,000× g for 15 min at 4 • C. Each test sample was combined equally to make a quality control (QC) sample. The analytical method for the QC sample was the same as for the test samples, and the QC sample was inserted after every six test samples to check the instrument's stability and performance.

Statistical Analysis
The data were analyzed by a one-way analysis of variance (ANOVA) using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Duncan's multiple range tests with significant differences of p < 0.05 were used to compare differences among the mean values. Origin (V. 2022.SR1) software (OriginLab, Northampton, MA, USA) was used to map volcano map of DMs, R language was used for principal component analysis (PCA) and mapped the bubble map of the pathway enrichment analysis.

Effects of Temperature on the BD, PPO, POD and PAL Enzyme Activities and MDA Content of Lanzhou Lily Bulbs
As shown in Figure 1 and Table 1, low-temperature preservation at 4 • C was beneficial to delay the rise of the BD of Lanzhou lily bulbs, inhibited the activities of related enzymes (PPO, POD, PAL) and browning, and maintained a low MDA content. Therefore, the low temperature of 4 • C can preserve the quality of Lanzhou lily bulbs throughout the storage period.

Effects of MAP on the BD, PPO, POD and PAL Enzyme Activities and MDA Content of Lanzhou Lily Bulbs
As shown in Figure 2 and Table 2, the results showed that 10% O 2 + 5% CO 2 + 85% N 2 (MA2) had the most obvious effect on inhibiting the activities of PPO, POD, PAL and the content of MDA, and the BD was the lowest under these conditions.

Sequencing Data Quality Assessment
By evaluating the transcriptome data of 24 samples (bx1, ck1 (1d), bx2, ck2 (4d), bx3, ck3 (8d), bx4, ck4 (14d)), RNA-Seq sequencing yielded a total of 43,541,142~55,338, 226 raw reads; Q20 was 97.39~97.78%, Q30 was 92.86~93.83%, and the GC content was 48.44~49.88%, after data filtering. Raw reads were 43,423,484~55,172,454, Q20 was 97.54~97.92%, Q30 was 93.04~94.01%, and the GC content was 48.47~49.93%. The sequencing error rate of each base position was less than 0.5%, the average quality peak value of most base sequences is greater than 30, therefore the sequencing quality met the standard, and further data analysis was performed. The detailed transcriptome data are in the attachment. There are certain differences in the expression of FPKM (fragments per kilobase of transcript per million reads mapped) of genes in each sample, but this difference is not obvious (Figure 3a,b). The correlation analysis (Pearson correlation) showed that the similarity of expression patterns between samples was high, and the samples' repeatability was good, the test was reliable, and the sample selection was reasonable. At the same time, it can be seen that there are differences in gene expression changes between the preservation treatment and the control group. Moreover, with the prolongation of keep-fresh time, the gene expression also changes ( Figure 3c). The results of PCA analysis showed that the Lanzhou lily bulb samples had obvious separation on the 1d, 4d, 8d and 14d, and the difference between the 4d and 8d were small, indicating that DEGs existed at all stages ( Figure 3d) (see data in Supplementary Material S1: Data quality assessment.xlsx).

Differentially Expressed Genes (DEGs) Analysis
We set principal standards of |log 2 (FoldChange)| > 1 and q value ≤ 0.05 to acquire DEGs that were significant from the dataset; the results showed that on the 1d (bx1 vs.

GO Enrichment Analysis of DEGs
GO enrichment analysis was performed on the DEGs ( Figure 5), which is used to study the functions of the DEGs at the three levels of Biological Process (BP), Cellular Component (CC) and Molecular Function (MF). On the 1d (Figure 5a), 10 GO terms such as 'metal ion binging' and 'sequence-specific DNA binding transcription factor activity' were mainly enriched at the MF level. At the CC level, 11 GO terms were mainly enriched such as 'nucleus, integral component of membrane', etc. A total of nine GO terms such as 'defense response and response to oxidative stress' were enriched at the BP level. On the 4d (Figure 5b), 16 GO terms such as 'ATP binding and RNA binding' were mainly enriched at the MF level. A total of seven GO terms such as 'integral component of membrane' were mainly enriched at the CC level. A total of seven GO terms such as 'transcription', 'DNA-templated' and 'cell wall organization' were enriched at the BP level. On the 8d (Figure 5c), nine GO terms such as 'ATP binding' and 'metal ion binding' were mainly enriched at the MF level. The CC level was mainly enriched with seven GO terms such as 'nucleus', 'integral component of membrane', 'cytoplasm', 'plasma membrane', etc. The BP level was enriched with 14 GO terms such as 'defense response'. On the 14d (Figure 5d

Differentially Expressed Genes (DEGs) Analysis
We set principal standards of |log2 (FoldChange)| > 1 and q value ≤ 0.05 to acquire DEGs that were significant from the dataset; the results showed that on the 1d (bx1 vs.     'integral component of membrane', 'cytoplasm', 'plasma membrane', etc. The BP level was enriched with 14 GO terms such as 'defense response'. On the 14d (Figure 5d), 10 GO terms such as 'ATP binding' and 'metal ion binding' were mainly enriched at the MF level. The CC level was mainly enriched with 10 GO terms such as 'nucleus', 'integral component of membrane', etc. A total of 10 GO terms such as 'defense response', 'regulation of transcription', 'DNA-templated', etc., were enriched at the BP level (see data in Supplementary Material S3: DEGs-GO analysis.xlsx).

Differential Metabolites (DMs) Analysis
The OPLS-DA model was used to evaluate the DMs, and the OPLS-DA scores show that each treatment group could be well separated from the control group, which indicated that the metabolites had undergone great changes. As shown in Figure 7, the results of the OPLS-DA model overfitting analysis (200 hypothesis tests) showed that the model was of good quality and was not overfitted. At the same time, VIP was used to identify the main contributing metabolites of the OPLS-DA model.

Differential Metabolites (DMs) Analysis
The OPLS-DA model was used to evaluate the DMs, and the OPLS-DA scores show that each treatment group could be well separated from the control group, which indicated that the metabolites had undergone great changes. As shown in Figure 7, the results of the OPLS-DA model overfitting analysis (200 hypothesis tests) showed that the model was of good quality and was not overfitted. At the same time, VIP was used to identify the main contributing metabolites of the OPLS-DA model. In univariate analysis-fold change analysis, DMs were screened with |log2 (FoldChange)| > 1 and p (adjust) value < 0.05. The results showed that on 1d (bx1 vs. ck1), 61 up-regulated DMs were identified in positive ion mode (Figure 8a (1)), and negative ion mode identified 1990 up-regulated and 882 down-regulated DMs (Figure 8a (2)). On 4d (bx2 vs. ck2), positive ion mode identified 372 up-regulated and 412 down-regulation DMs (Figure 8b (1)), and negative ion mode identified 587 and 891 DMs that were upregulated and down-regulated (Figure 8b (2)). On 8d (bx3 vs. ck3), positive ion mode identified 227 and 915 up-regulated and down-regulated DMs (Figure 8c (1)), and

KEGG Pathway of Differential Gene and Metabolite Co-Enrichment Analysis
To further explore the interaction between DEGs and DMs, we performed a coenrichment analysis of transcriptome and metabolome. As shown in Figure 10, on the 1d, there were 8 enriched pathways for DEGs and DMs, mainly including 'Porphyrin and chlorophyll metabolism' and 'Nitrogen metabolism'. On the 4d, there were 11 enriched pathways, mainly including 'Phenylpropanoid biosynthesis' and 'Linoleic acid metabolism'. On the 8d, a total of 13 pathways were enriched, mainly including 'Phenylpropanoid biosynthesis', 'Linoleic acid metabolism' and 'alpha-Linoleic acid metabolism'. On the 14d, there were a total of enriched 15 pathways, mainly including 'Phenylpropanoid biosynthesis', 'Linoleic acid metabolism' and 'alpha-Linolenic acid metabolism' (Figure 10).

Changes of Metabolites and Genes
The DEGs and DMs involved in the browning of Lanzhou lily bulbs mainly affected 'Phenylpropanoid biosynthesis', 'Flavonoid biosynthesis' and 'Stilbenoid, diarylheptanoid and gingerol biosynthesis'. Lipid metabolism, including 'linoleic acid metabolism' and 'alpha-linolenic acid metabolism', also were affected. On the 1d, the genes involved in the phenylpropane pathway, such as PAL, cinnamoyl alcohol dehydrogenase (CAD), Cinnamic acid 4-hydroxylase (C4H), Peroxidases, Chalcone synthase (CHS) and Chalcone reductase (CHR) were up-regulated. On the 14d, the main genes involved in the phenylpropane pathway were almost all down-regulated, and genes in the linoleic acid metabolism and alpha-linolenic acid metabolism were also down-regulated (Figures 11 and 12).

KEGG Pathway of Differential Gene and Metabolite Co-Enrichment Analysis
To further explore the interaction between DEGs and DMs, we performed a c enrichment analysis of transcriptome and metabolome. As shown in Figure 10, on the 1 there were 8 enriched pathways for DEGs and DMs, mainly including 'Porphyrin an chlorophyll metabolism' and 'Nitrogen metabolism'. On the 4d, there were 11 enriche pathways, mainly including 'Phenylpropanoid biosynthesis' and 'Linoleic ac metabolism'. On the 8d, a total of 13 pathways were enriched, mainly includin 'Phenylpropanoid biosynthesis', 'Linoleic acid metabolism' and 'alpha-Linoleic ac metabolism'. On the 14d, there were a total of enriched 15 pathways, mainly includin 'Phenylpropanoid biosynthesis', 'Linoleic acid metabolism' and 'alpha-Linolenic ac metabolism' (Figure 10).  metabolism' and 'alpha-linolenic acid metabolism', also were affected. On the 1d, the genes involved in the phenylpropane pathway, such as PAL, cinnamoyl alcohol dehydrogenase (CAD), Cinnamic acid 4-hydroxylase (C4H), Peroxidases, Chalcone synthase (CHS) and Chalcone reductase (CHR) were up-regulated. On the 14d, the main genes involved in the phenylpropane pathway were almost all down-regulated, and genes in the linoleic acid metabolism and alpha-linolenic acid metabolism were also downregulated (Figures 11 and 12).

Discussion
Appearance and texture are two fundamental factors that determine the acceptability of FFV [22]. Browning and softening during post-harvest storage cause the appearance quality of Lanzhou lily bulbs to decline, thus affecting consumers' acceptability and purchasing power. Mechanical damage (fresh cutting) triggers increased respiratory rate and ethylene production in plant tissues [23], destruction of membrane integrity [24] and a large number of physiological and biochemical reactions such as the biosynthesis of secondary metabolites [25].
The integrity of the cell membrane is one of the key factors affecting the browning of fruits and vegetables, mechanical-damage-enhanced membrane lipid peroxidation and lost membrane integrity [26]. Lipids are the basic components of cell membranes. The escape of phenolic substances produces a browning of the tissues under the pro-browning action of Polyphenol oxidase (PPO) [27]. Studies show that linolenic acid and linoleic acid play important roles in fruit browning [28], especially in reducing the proportion of unsaturated fatty acids, which result in the browning of fruits and vegetables [29]. A delayed reduction in linoleic acid and linoleic acid content and increased proportion of unsaturated fatty acid/saturated fatty acid in the fruit can help reduce browning inside the fruit [30,31]. Transcriptomics and metabolomics data showed that key enzymes for the synthesis of linolenic acid and linoleic acid, Phospholipase A2 Group (PLA2G), were up-regulated later in MAP. Linolenic acid and linoleic acid are the main substrates of Lipoxygenase (LOX), so when the activity of lipoxygenase LOX is low, and the metabolic pathway downstream of linolenic acid and linoleic acid metabolism is inhibited, high levels of linolenic acid and linoleic acid are maintained, which inhibits browning. Saquet et al. [32] pointed out that after 'Conference' pears were treated by MAP, the relative content of unsaturated fats (such as linolenic acid and linoleic acid) were maintained, the integrity of cell membranes was maintained, and the browning reaction was inhibited. Lin et al. [33] found that the peel of Longan (Dimocarpus longan Lour.) treated with hydrogen peroxide (H 2 O 2 ) had a higher browning index, higher LOX activity and a lower relative content ratio of unsaturated fatty acids (linoleic acid, linolenic acid)/saturated fatty acids. The cell membrane permeability increases, and the integrity of the membrane structure is lost. MDA can be used as a biochemical marker of membrane structural integrity. LOX catalyzes the oxidation of polyunsaturated fatty acids to conjugated diene and malondialdehyde (MDA); once accumulated, MDA can further damage cell membranes. Dhindsa et al. [34] Gao et al. [35] and Jiang et al. [36] found that the integrity of the membrane was lost during the browning process of fresh-cut lotus root in the control group, and the content of MDA increased in his browning model study. Dokhanieh et al. [37] found that after fresh-cut pomegranate arils were stored at 4 • C for 12 d, browning occurred simultaneously with the accumulation of MDA and H 2 O 2 and, after hot salicylic acid treatment, the accumulation of MDA was reduced and browning was delayed, which is consistent with our experimental results in this study showing that the MAP reduced the MDA accumulation in Lanzhou lily bulbs. To sum up, this indicates that the MAP may inhibit the browning of Lanzhou lily bulbs by delaying the decrease of the ratio of unsaturated fatty acid/saturated fatty acid content in the cell membrane, further inhibiting membrane lipid peroxidation and maintaining the integrity of the cell membrane of the lily bulbs.
Notably, the biosynthesis of secondary metabolites is another important factor affecting the browning of fruits and vegetables, among which phenylpropane metabolism is an important pathway [38]. Phospholipase (PAL) is an important enzyme of the phenylpropane pathway, catalyzing the formation of L-phenylalanine to trans-cinnamic acid, and then, through a series of biochemical reactions to generate various phenolic substances, anthocyanins, lignin, alkaloids and other substances [39,40], these metabolites provide a precursor substance for oxidative browning. Usually after the plant epidermis is broken (freshly cut), PAL is expressed in large quantities. After the integrity of the cell membrane is impaired, phenolic substances in the vacuole leak, browning-related enzymes and substrates come into contact with each other, and phenolic substrates oxidize to form browning products [41]. In addition, PPO is closely related to tissue browning in fruits and vegetables [42], because PPO can oxidize phenols to quinones under O 2 catalysis, forming browning products [43]. PPO oxidizes phenols to produce H 2 O 2 , then uses H 2 O 2 as an electron acceptor to catalyze the oxidation of phenols to form browning products [44]. Physiological experiments have shown that PAL activity, PPO activity and POD activity were at lower levels in the late stage of MAP compared with the control; these results indicate that MAP may inhibit the oxidation reaction of phenolic substances leaking from fresh-cut lily bulbs by inhibiting the activity of PAL, PPO and POD, and further inhibit the formation of browning products. This is similar to the conclusions of numerous studies [41]. High-pressure carbon dioxide (HPCD) treatment of fresh-cut lettuce has shown that HPCD can effectively inhibit PPO and PAL activity [45]. Eugenol emulsions (EUG) on fresh-cut Chinese water chestnut (CWC) showed that EUG reduced the content of phenols and inhibited the browning reaction by reducing the activity of PPO, POD and, especially, PAL enzymes. It is worth noting that phenylpropanoid biosynthesis, flavonoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis are closely related to post-harvest disease resistance and color formation of fruits and vegetables. Studies have shown that key genes in the 'carotenoid biosynthesis' pathway, 'flavonoid biosynthesis' pathway and 'stilbenoid, diarylheptanoid and gingerol biosynthesis' pathway in fresh-cut yam are significantly upregulated during yellowing after storage [46]; in the fresh-cut easy-to-brown potato variety YS505, the phenylpropanoid biosynthesis pathway was activated [47]. In addition, it is common for mechanical damage to induce the synthesis and accumulation of phenolic compounds in FFV [48,49]; in order to defend and heal damage, plants quickly synthesize phenolic substances, especially phenolic anti-oxidants, in a short period of time [50]. Many studies have also shown that after damage to different types of freshly cut fruits and vegetables, reactive oxygen species (ROS) were produced rapidly and the content of phenolic compounds and anti-oxidant activity is significantly increased [51].
In this study, omics results showed that the key genes PAL, CAD, C4H Peroxidases, CHS and CHR in phenylpropanoid biosynthesis, flavonoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis were up-regulated in the early stage of MAP. There is another possible reason that, compared with the air control, the MAP group gas composition ratio changes, further activating the stress response and enhancing the synthesis pathway of phenolic compounds, but there are few studies in this regard, and the specific mechanism remains to be discussed. It is assuring that the key genes of phenylpropanoid biosynthesis, flavonoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis were down-regulated in the post-MAP period (14 d). Studies have shown that low and high O 2 /CO 2 atmospheres can reduce the respiration rate and browning of freshcut potatoes [52]. Similarly, high-pressure carbon dioxide (HPCD) treatment displayed low phenylpropanoid metabolism pathway activity in fresh-cut Chinese water chestnut (CWC) [53]. From this, we speculate that MAP inhibits the synthetic accumulation of phenols and the browning caused by their oxidation in the fresh-cut Lanzhou lily bulb.
The mechanism of anti-browning MAP technology in the preservation of Lanzhou lily bulbs was preliminarily studied through transcriptomics and metabolomics data, and the key genes affecting browning and the key secondary metabolites produced during browning were found. The signal regulatory pathways related to anti-browning were also revealed in this study. However, the continuation of this research needs to verify the anti-browning mechanism of key genes using more molecular biology technology, and verify key secondary metabolites by targeted metabolomics and molecular biology technology, so as to better reveal the anti-browning mechanism of MAP in the preservation process of Lanzhou lily bulbs. This study provided theoretical value and data support for the preservation of fresh-cut fruits and vegetables.

1.
Fresh-cut Lanzhou lily bulbs have the lowest browning index, a good quality and good appearance under the conditions of MAP of 10% O 2 + 5% CO 2 + 85% N 2 and 4 • C. 2.
MAP reduces the activity of PAL, PPO, POD and the content of MDA. 3.
The mechanism by which MAP inhibits the browning of fresh-cut Lanzhou lily bulbs may be that it retards the reduction in the ratio of unsaturated fatty acids to saturated fatty acids in the cell membrane of the bulbs. Specifically, MAP inhibits the lipid peroxidation of the membrane to maintain the integrity of the cell membrane, and probably inhibits the metabolic pathways of 'Phenylpropanoid biosynthesis', 'Flavonoid biosynthesis' and 'Stilbenoid, diarylheptanoid and gingerol biosynthesis' and the expression of their key enzyme genes, thus inhibiting the oxidation of phenolic substances.

Data Availability Statement:
The data used to support the findings of this study can be made available by the corresponding author upon request.

Conflicts of Interest:
The authors declare no conflict of interest.